It is increasingly clear that proteins of the extracellular matrix (ECM) are key regulators of cell patterning and morphogenesis in the developing nervous system. Growth factors and morphogens, such as fibroblast growth factors (Fgfs), sonic hedgehog (Shh) and retinoic acid (RA), are critical regulators of nervous system development. The present proposal continues our analysis of the effects of ethanol exposure during embryogenesis on ECM function, in particular agrin Shh, Fgfs and RA. Our overall goal is to understand how gene-ethanol interactions contribute to the hallmark features of fetal alcohol spectrum disorders (FASD). A focus of this proposal is to employ a new multiple binge-like alcohol exposure model during defined periods of zebrafish and mouse development to identify critical stages that are sensitive to ethanol exposure and that lead to the morphological and behavioral changes associated with FASD. The zebrafish model offers a unique advantage in that following ethanol removal the tissue levels of ethanol rapidly return to control levels, allowing defined exposures during embryogenesis. Thus, our underlying hypothesis is that gene-ethanol interactions involving agrin, Fgfs, Shh and RA during defined stages of embryogenesis are necessary to produce the hallmark morphological features of FASD, and the behavioral deficits that are characteristic of FASD. Our proposed studies take the innovative approach of using multiple subthreshold binge ethanol exposures during embryogenesis to reconcile the disparity between reported alcohol use during pregnancy and the incidence of FAS or FASD in the population. The specific aims of this proposal are: 1) To test the hypothesis that multiple binge-like alcohol exposures during defined stages of zebrafish or mouse embryogenesis produce characteristic morphological FASD phenotypes and associated gene expression alterations; 2) To test the hypothesis that multiple binge-like alcohol exposures during defined stages of zebrafish or mouse embryogenesis lead to behavioral changes associated with FASD in humans. These Specific Aims will further establish that pathological and behavioral phenotypes associated with FASD are regulated by the timing of ethanol exposure and the underlying gene-ethanol interaction, and will elucidate the role of ECM ligands such as agrin, Shh, Fgfs and RA in the ethanol-induced morphological and behavioral defects associated with these binge-like alcohol exposures.